Methods for reducing matrix-bound nicotine-derived nitrosamine ketone in tobacco plant material

ABSTRACT

A method of reducing the amount of matrix-bound NNK in cured tobacco plant material is provided comprising reducing the amount of lignin in the cured tobacco plant material. A further method of reducing the formation of matrix-bound NNK during the curing of tobacco plant material is described comprising reducing the amount of lignin therein prior to curing.

FIELD OF THE INVENTION

The present invention relates, in general, to methods for reducing theamount of nicotine-derived nitrosamine ketone or4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco plantmaterial.

BACKGROUND OF THE INVENTION

During the manufacture and processing of tobacco products,by-products—such as tobacco stems, and leaf scraps—are produced. Tobaccostems and tobacco fines from manufacturing processes are unsuitable foruse directly in the manufacturing of tobacco products. Since the stemsand fines represent a substantial amount of raw material investment,processes have been developed to further convert these stems and finesinto products—such as reconstituted tobacco materials (eg. reconstitutedtobacco sheets)—which are then useable in relatively large amounts in amixture with acceptable processed tobacco leaf. Reconstituted tobaccocan be manufactured in a slurry or cast sheet process wherein pulp ofmashed tobacco stems and other parts of the tobacco leaf are ground andmixed with a solution that might contain different additives. Theresulting tobacco slurry is then sprayed to form a thin film, dried,rolled and diced into strips which are added to a filler. Nitrosaminesare organic compounds found in many consumer products—such as tobacco,food products and cosmetics. Nitrosamines have drawn intense scientificinterest because some of the compounds in this class have been shown tobe carcinogenic in laboratory animals. It has been reported that somecured tobaccos contain tobacco specific nitrosamines that can be foundin smokeless tobacco, mainstream smoke and side stream smoke ofcigarettes. In tobacco, at least four species of nitrosamines areproduced at appreciable quantity. These are nicotine-derived nitrosamineketone or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), andN-nitrosoanabasine (NAB). Tobacco specific nitrosamines are notconsidered to be present in significant quantities in growing tobaccoplants or fresh cut tobacco (green tobacco), but are formed during thetobacco curing process. In addition to the formation of tobacco specificnitrosamines during the curing process of green leaves, tobacco specificnitrosamines may also be formed during processes used to prepare aqueoustobacco slurries—such as processes used to prepare reconstitutedtobacco.

In an attempt to reduce tobacco specific nitrosamines, varioustreatments of tobacco plants or harvested tobacco leaves have beensuggested, including radiation treatments, chemical treatments andextractions. Other methods for reducing tobacco specific nitrosamineshave been suggested by MacKown et al. (1988) J. Agric. Food Chem. 36,1031-1035. These methods involve treatment using sterilization,microbial inhibitors, bases to increase pH, or ascorbic acid to decreasethe accumulation of tobacco specific nitrosamines during the productionof reconstituted tobacco sheets. WO2012160133 describes a process fordecreasing the levels of tobacco specific nitrosamines in tobaccohomogenates by increasing the pH thereof, especially when elevatedlevels of nitrosamines are created by elevated nitrite levels.

One problem with trying to reduce the levels of tobacco specificnitrosamines in tobacco is that some of the nitrosamines in air-curedtobacco, including NNK, exist in a bound or matrix-bound form, which canbe difficult to remove or extract. Matrix-bound NNK can be extractedwith a 0.1 N KOH solution from water-washed Burley filler. This alkalinetreatment also decreases NNK levels in smoke (Keene, C. K., 1992, TheEffect of Base Digestion on TSNA in Extractables-Depleted Fillers.Legacy Tobacco Documents). However, the treatment can introducetoxicologically relevant compounds into tobacco and can significantlydeteriorate the quality of the tobacco, which is highly problematic forthe tobacco industry.

WO2010/021809 describes a method for recuing nitrogen compounds andlignin in tobacco. Nitrogen compounds are removed by solvent extraction;lignin is removed in a separate step.

A need remains for an effective and cost efficient method for reducingmatrix-bound NNK that is formed during the curing of tobacco. Inparticular, a cost-effective and simple method for reducing the levelsof matrix-bound NNK in cured tobacco that does not introduce toxic orpotentially toxic compounds and does not deteriorate the quality of thetobacco product is particularly desirable.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the surprisingfinding that matrix-bound NNK co-localises with lignin in tobaccoplants. In particular, the present inventors have observed that withincured parts of a tobacco plant—such as in the stems andmidribs—matrix-bound NNK co-localises or co-localises predominantly orco-localises exclusively with lignin—such as lignified tissue,particularly in the vascular bundle (for example, the xylem) and not thesurrounding tissues—such as the cortex. Therefore removal of lignin (forexample, by separating lignified from non-lignified tissue) can reducethe amount of matrix-bound NNK and metabolites thereof in plantmaterial. Matrix-bound NNK can be covalently or non-covalently linked tolignin. The plant material is expected to deliver smoke with reduced NNKlevels and potentially improved sensory properties. The presentdisclosure can be applied to those plant materials which accumulatematrix-bound NNK or have the potential to accumulate matrix-bound NNK.In particular, the disclosure can be applied to low-value plant materialcomprising matrix-bound NNK that is used in certain tobacco processes.The methods described herein can be carried out without the use of anyadditives and thereby do not introduce additional toxicologicallyrelevant compounds into the plant material. The removal of lignin cantake place during or after curing of tobacco plant material. Lignin canbe removed before curing to prevent, reduce or inhibit matrix-bound NNKco-localising with lignin.

One general object of this disclosure is to substantially decrease theamount of matrix-bound NNK and metabolites thereof in tobacco intendedfor smoking or consumption by other means. Another general object is toreduce the carcinogenic potential of tobacco products, includingcigarettes, cigars, chewing tobacco, snuff and tobacco-containing gumand lozenges. Still another general object is to decrease or reduce theamount of matrix-bound NNK and metabolites thereof in tobacco plantmaterial and in tobacco products. Another general object is to reducethe amount of matrix-bound NNK and metabolites thereof in cured—such aspartially or fully cured—tobacco plant material. Another general objectis to reduce the amount of NNK and metabolites thereof in aerosol,including smoke. Yet another object of this disclosure is to reduce theamount of NNK or metabolites thereof in humans who smoke, consume orotherwise ingest tobacco in some form, by providing a tobacco productsuitable for human consumption which contains a reduced amount of NNK ormetabolites thereof, thereby lowering the carcinogenic potential of suchproduct.

In one aspect, there is provided a method of reducing the amount ofmatrix-bound NNK in a cured tobacco plant or in cured tobacco plantmaterial comprising reducing the amount of lignin therein by separatinglignified from non-lignified tissue, preferably, wherein the amount oflignin is reduced chemically and/or mechanically.

In one embodiment, the tobacco plant or the tobacco plant material istreated to expand non-lignified plant tissue. The amount of lignifiedtissue is reduced by separating the expanded and non-expanded planttissue based on their different densities (for example, buoyantdensities) and/or their different strengths and/or their different sizesand/or their different weight. The expanded plant tissue can becollected for further tobacco processing.

It has been observed that significant reduction in levels of NNK andassociated TSNAs can be obtained by fractionating the lignified tissues.Separate chemical extraction of nitrogenous compounds and/or lignin isnot necessary.

In one embodiment, the amount of lignin is reduced by removing thevascular bundle or xylem or lignified sclerenchymatic tissue or acombination of two or more thereof from the plant or plant material. Thelignin can be located in the vascular bundle. The lignin can be locatedexclusively in the vascular bundle. The lignin can be locatedexclusively in the vascular bundle and not the surrounding tissue. Thelignin can be located in the xylem. The lignin can be locatedexclusively in the xylem. The lignin can be located exclusively in thexylem and not the surrounding tissue. The lignin can be located insclerenchymatic tissue. The lignin can be located exclusively insclerenchymatic tissue. The lignin can be located exclusively in thesclerenchymatic tissue and not the surrounding tissue. Lignified tissueis generally absent from the outer layer of plant midribs.

In one embodiment, the plant or plant material that is treated accordingto the present disclosure comprises or consists or consists essentiallyof plant midribs or plant stems or plant stalks or a combination of twoor more thereof.

In one embodiment, the amount of lignin is reduced by harvesting thecortex—such as the outer cortex—from the plant or plant material.

In one embodiment, the method comprises the steps of: (a) providing acured tobacco plant or cured tobacco plant material; (b) reducing theamount of lignin in the cured tobacco plant or the cured tobacco plantmaterial by fractionating the tobacco plant material; and (c) obtaininga cured tobacco plant or cured tobacco plant material in which theamount of lignin is reduced and the amount of matrix-bound NNK isreduced as compared to the cured tobacco plant or the cured tobaccoplant material provided in step (a).

In one embodiment, following step (a) there is a further step ofmeasuring the amount of free NNK or matrix-bound NNK or a combinationthereof, and optionally, wherein following step (b) there is a furtherstep of measuring the amount of free NNK or matrix-bound NNK or acombination thereof. In one embodiment, the method comprises the furtherstep (d) of comparing the level of at least matrix-bound NNK measuredfollowing step (a) with the level of NNK measured following step (b),wherein a reduction in the amount of matrix-bound NNK in the tobaccomaterial obtained in step (b) as compared to the tobacco materialprovided in step (a) is indicative that the amount of matrix-bound NNKin the tobacco material is reduced.

In a further aspect, there is provided a method of reducing theformation of matrix-bound NNK during the curing of a tobacco plant ortobacco plant material comprising reducing the amount of lignin thereinprior to curing.

In one embodiment, the method comprises the steps of: (a) providing anuncured tobacco plant or uncured tobacco plant material; (b) reducingthe amount of lignin in the uncured tobacco plant or the uncured tobaccoplant material prior to curing; (c) curing the tobacco plant or thetobacco plant material provided in step (b); and (d) obtaining a curedtobacco plant or cured tobacco plant material in which the amount ofmatrix-bound NNK is reduced as compared to a control in which the amountof lignin has not been reduced.

In one embodiment, following step (a) there is a further step ofmeasuring the amount of free NNK or matrix-bound NNK or a combinationthereof, and optionally, wherein following step (b) there is a furtherstep of measuring the amount of free NNK or matrix-bound NNK or acombination thereof and optionally, wherein following step (c) there isa further step of measuring the amount of free NNK or matrix-bound NNKor a combination thereof.

In one embodiment, following step (c) or step (d) said method comprisesthe further step of comparing the level of at least matrix-bound NNKmeasured following step (a) with the level of NNK measured followingstep (b) and/or step (c), wherein a reduction in the amount ofmatrix-bound NNK in the tobacco material obtained in step (b) or step(c) as compared to the tobacco material provided in step (a) isindicative that the amount of matrix-bound NNK in the tobacco materialis reduced.

In a further aspect, there is provided tobacco plant material obtainedor obtainable by the method(s) described herein.

In a further aspect, there is provided the use of a tobacco plant ortobacco plant material in which the amount of lignin therein has beenreduced as compared to control tobacco plant or control tobacco plantmaterial for manufacturing tobacco with reduced levels of matrix-boundNNK, wherein said levels of matrix-bound NNK are reduced as compared tothe control.

In a further aspect, there is provided a method for producingreconstituted tobacco comprising the steps of: (a) performing themethod(s) described herein; (b) manufacturing the tobacco materialobtained in step (a) into reconstituted tobacco; and (c) optionallyincorporating the reconstituted tobacco into a tobacco product.

In a further aspect, there is provided reconstituted tobacco obtained orobtainable by the method described herein.

In a further aspect, there is provided a method for preparing tobaccofor use as a tobacco cut filler comprising the steps of: (a) performingthe method(s) described herein; and (b) rolling and cutting the tobaccomaterial for use as a tobacco cut filler.

In a further aspect, there is provided cured tobacco plant materialcontaining a reduced level of lignin as compared to control tobaccoplant material in which the amount of lignin has not been reduced, andwherein the amount of matrix-bound NNK is about 3500 ng/g or less.

In one embodiment, the average particle size is greater than about 0.5millimetres.

In one embodiment, the amount of free NNK is less than about 330 ng/g,optionally wherein the NNN content is less than about 1700 ng/g andoptionally wherein the nicotine content is less than about 2610 μg/g.

In one embodiment, the cured tobacco plant material comprises, consistsof consists essentially of plant cortex—such as outer plant cortex.

In one embodiment, vascular bundle or xylem or lignified sclerenchymatictissue or a combination of two or more thereof is substantially absentfrom the cured tobacco plant material.

In one embodiment, the cured tobacco plant material comprises, consistsof consists essentially of plant cortex—such as outer plant cortex—andvascular bundle or xylem or lignified sclerenchymatic tissue or acombination of two or more thereof is substantially absent therefrom.

In one embodiment, the cured tobacco plant material is obtained orobtainable from plant midribs or plant stems or plant stalks or acombination of two or more thereof.

In one embodiment, the average particle size is greater than about 0.5millimetres.

In one embodiment, the amount of free NNK is less than about 330 ng/g.

In one embodiment, the NNN content is less than about 1700 ng/g.

In one embodiment, the nicotine content is less than about 2610 μg/g.

In a further aspect, there is provided a tobacco product or areconstituted tobacco product comprising, consisting or consistingessentially of the plant material or the cured plant material describedherein.

In a further aspect, there is provided a method of producing an aerosolin which the amount of NNK is reduced as compared to a control aerosolcomprising the steps of: (a) providing a cured tobacco plant or curedtobacco plant material; (b) reducing the amount of lignin in the curedtobacco plant or the cured tobacco plant material; (c) obtaining a curedtobacco plant or cured tobacco plant material in which the amount oflignin is reduced and the amount of matrix-bound NNK is reduced ascompared to the cured tobacco plant or the cured tobacco plant materialprovided in step (a); and (d) heating the cured tobacco plant or curedtobacco plant material from step (c) to produce an aerosol.

In a further aspect, there is provided a method of producing an aerosolin which the amount of NNK is reduced as compared to a control aerosolcomprising the steps of: (a) providing an uncured tobacco plant oruncured tobacco plant material; (b) reducing the amount of lignin in theuncured tobacco plant or the uncured tobacco plant material prior tocuring; (c) curing the tobacco plant or the tobacco plant materialprovided in step (b); (d) obtaining a cured tobacco plant or curedtobacco plant material in which the amount of matrix-bound NNK isreduced as compared to a control in which the amount of lignin has notbeen reduced; and (e) heating the cured tobacco plant or cured tobaccoplant material from step (d) to produce an aerosol.

In a further aspect, there is provided a method of producing an aerosolin which the amount of NNK is reduced as compared to a control aerosolcomprising the step of: (a) providing a tobacco product or areconstituted tobacco product comprising, consisting or consistingessentially of the tobacco plant material or the cured plant materialobtained or obtainable by the methods described herein; and (b) heatingthe tobacco product or the reconstituted tobacco product to produce anaerosol.

In a further aspect, there is provided an aerosol obtained or obtainableby the method(s) described herein.

In a further aspect, there is provided cured tobacco plant materialconsisting essentially of tobacco plant cortex and wherein the amount ofmatrix-bound NNK is reduced as described herein.

In a further aspect, there is provided a method for blending tobacco inwhich at least two different types of tobacco are blended so as to forma tobacco blend comprising the steps of: (a) providing a first curedtobacco plant material and reducing the amount of lignin therein; (b)measuring the total and/or matrix-bound NNK content of the first curedtobacco plant material and selecting cured tobacco plant material inwhich the total and/or matrix-bound NNK content is reduced as comparedto the first cured tobacco plant material provided in step (a); (c)providing a second cured tobacco plant material which has a higher totaland/or matrix-bound NNK content than the total and/or matrix-bound NNKof the first cured tobacco plant material obtained in step (b), andoptionally measuring the total and/or matrix-bound NNK content in thesecond cured tobacco plant material; (d) blending together the first andsecond cured tobacco plant materials from steps (b) and (c) andoptionally measuring the total and/or matrix-bound NNK content in theblended tobacco plant material; and (e) obtaining a blended tobaccoplant material in which the total and/or matrix-bound NNK content of theblended tobacco plant material is lower than the second cured tobaccoplant material provided in step (c), optionally wherein steps (a) and(b) are performed after step (c).

In a further aspect, there is provided blended tobacco plant materialobtained or obtainable by the method(s) described herein.

Each of the embodiments discussed above are disclosed as embodiments ofeach of the aspects of the invention. Combinations of one or of theembodiments are contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the distribution of free NNK,matrix-bound NNK and lignin in lignified (L) and non-lignified (NL)tissues in cured Burley midribs or cured Burley stems.

FIG. 2 is a cross-section of a hydrated cured Burley stem showinglignified (L) and non-lignified (NL) tissues. Lignified tissue isstained red with phloroglucinol.

FIG. 3 is a graph illustrating the distribution of free NNK,matrix-bound NNK and lignin in lignified (L) and non-lignified (NL)tissues of green Burley midribs after nitrosation with sodium nitritesolution (1.5 mL (10 mg/mL in water) for 4 hours at room temperaturewith shaking).

FIG. 4 is a graph illustrating free and matrix-bound NNK in sievingfractions of ground freeze-dried Burley tobacco plant stems.

FIG. 5 is graph showing the correlation between matrix-bound NNK andlignin in sieving fractions of ground freeze-dried Burley tobacco plantstems.

FIG. 6 is a graph showing the concentration of matrix-bound NNK (μg/g)in sclerenchymatic tissue (S) and in the outer layers of the midribs(NS) after nitrosation and washing in green midribs of TN90. Levels ofpseudo-oxynictoine (PON) (μg/g) and nicotine (μg/g) are also shown.Matrix-bound NNK levels (ng/g) in lignified (CS) and non-lignified (CNS)parts of a commercial cured Burley stem sample are also shown. Levels ofNNN (ng/g) and nicotine (μg/g) are also shown.

DEFINITIONS

The technical terms and expressions used herein are generally to begiven the meaning commonly applied to them in the pertinent art of plantand molecular biology. All of the following term definitions apply tothe complete content of this disclosure.

The word “comprising” does not exclude other elements or steps, and theindefinite article “a” or “an” does not exclude a plurality.

The terms “essentially”, “about”, “approximately” and the like inconnection with an attribute or a value particularly also define exactlythe attribute or exactly the value, respectively.

The term “plant” refers to any plant or any part thereof at any stage ofits life cycle or development, and its progenies. In one embodiment, theplant is a tobacco plant, which refers to a plant belonging to the genusNicotiana. Preferred species of tobacco plant are described herein.

A “plant cell” refers to a structural and physiological unit of a plant.The plant cell may be in the form of a protoplast without a cell wall,an isolated single cell or a cultured cell, or as a part of higherorganized unit such as but not limited to, plant tissue, a plant organ,or a whole plant. In one embodiment, the plant cell is a tobacco plantcell.

The term “plant material” refers to any part of a plant or a mixture ofdifferent parts of plant or a mixture of different plants and includeswithout limitation plant tissues, leaf scraps, green leaf scraps, stems,dust created during plant processing, and leaf prime lamina strip andcombinations thereof. In certain embodiments, the plant material willcomprise, consist or consist essentially of a plant part or a mixture ofplant parts containing lignin—such as plant midribs or plant stems orplant stalks or a combination or two or more thereof. Tobacco plantmaterial can have the form of processed tobacco parts or pieces,uncured, cured or aged tobacco in essentially natural lamina or stemform, a tobacco extract or a mixture of the foregoing, for example, amixture that combines extracted tobacco pulp with granulated cured andaged natural tobacco lamina. The plant material can be in solid form, inliquid form, in semi-solid form, in ground form, in crushed form, insieved form, or in particulate form or the like or otherwise treated toreduce particle size. The plant material can be in the form of ahomogenate that has been subjected to homogenization, including, but notlimited to cutting or grinding or a combination thereof. The homogenatemay be prepared from whole plants or from mixtures of plantcomponents—such as a mixture of plant parts containing lignin, forexample, midribs or stems or stalks or a combination of two or morethereof—that have been subjected to homogenisation. The plant materialcan be in the form of a slurry, including a suspension of plant materialor a plant homogenate in an aqueous solution or solvent. The slurry canbe a 5% (w/v), 10% (w/v), 15% (w/v), 20% (w/v) or 25% (w/v) or moremixture of plant material in an aqueous solution or solvent. In oneembodiment, the plant material is that plant material which comprises,consists or consists essentially of lignin—such as lignified tissue. Inone embodiment, the plant material is that plant material whichcomprises, consists or consists essentially of the vascular bundle. Inone embodiment, the plant material is that plant material whichcomprises, consists or consists essentially of xylem. In one embodiment,the plant material is that plant material which comprises, consists orconsists essentially of lignified sclerenchymatic tissue. In oneembodiment, the plant material comprises, consists or consistsessentially of plant midribs or plant stems or plant stalks or acombination of two or more thereof. In one embodiment, the plantmaterial is tobacco plant material.

The term “tobacco product” includes smoking or smokable articles, andsmokeless tobacco products.

The term “free NNK” refers to the NNK concentration calculated from theNNK content of extracts prepared by extracting said plant material withaqueous buffer(s) at room temperature. The free NNK content in suchextracts can be determined using ultra performance liquidchromatography-tandem mass spectrometry (UPLC-MS/MS).

The term “total NNK” refers to the NNK concentration calculated aftersubjecting the extraction mixtures to the methods described herein (forexample, by heating to about 130° C. for about 4 hours) and filteringaliquots of the extracts. The total NNK content in such extracts can bedetermined using UPLC-MS/MS.

The term “bound NNK” or “matrix-bound NNK” as used herein represents thedifference between the “total NNK” and the “free NNK” concentration.

The terms “reduced lignin content” or “decreased lignin content” or“non-lignified” grammatical variations thereof refers to a measurablequantitative reduction in the amount of lignin in a plant when comparedto the amount of lignin in a comparable control plant. A quantitativereduction of lignin can be readily ascertained by assays that are knownin the art and include the Klason lignin assay (Method in Enzymol.,161:87-101 (1988)), the acetyl bromide assay (Wood Sci. Technol.,22:271-280 1988)) or the photometric method based on derivatisation withthioglycolic acid (J. Chem. Ecol., 28, 2483-2501 (2002)). Innon-lignified tissue, the amount of lignin is decreased as compared to acomparable control plant and the amount of lignin can be completely,substantially or partially removed. In non-lignified tissue, adetectable amount of lignin can be present provided that there is ameasurable quantitative reduction in the amount of lignin when comparedto a comparable control plant in which the amount of lignin has not beenreduced. In non-lignified tissue it may not be possible to detect anyamount of lignin. Non-lignified tissue can contain less than 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total dry weight content oflignin in the plant or plant part from which it is removed.

A “control plant” or “control plant cell” refers to a plant or a plantcell—such as a native or naturally occurring plant or plant cell—havinga lignin content and/or a NNK content that has not been manipulated ormodified. Control plant material includes plant material obtained from,derived from or derivable from the control plant or the control plantcell or a combination thereof. The control plant or control plant cellcan be the same type of plant or plant cell, for example, the samespecies of plant or plant cell as the plant or plant cell that it isbeing compared to. The control plant or control plant cell maycorrespond to a wild-type plant or wild-type plant cell.

The term “reduce” or “decrease” or grammatical variations thereof refersto a reduction of from about 10% to about 99%, or a reduction of atleast 10%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 100% ormore of a quantity, amount or activity.

The term “inhibit” or grammatical variations thereof, refers to areduction of from about 98% to about 100%, or a reduction of at least98%, at least 99%, but particularly of 100%, of a quantity, amount oractivity.

The term “increase” or grammatical variations thereof refers to anincrease of from about 5% to about 99%, or an increase of at least 5%,at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 100% ormore of a quantity, amount or activity.

The term “about” in the context of a given numerate value or rangerefers to a value or range that is within 20%, within 10%, or within 5%of the given value or range.

The term “at least a portion” or grammatical variations thereof includesat least about 5%, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 90%, at least about 95%, at least about 98%, or at least about 99%of a quantity, amount or activity.

DETAILED DESCRIPTION

Generally speaking, the present disclosure can be applied to any form oftobacco plant material in which NNK or metabolites thereof or acombination thereof can form or have formed. Suitably, at least aportion of the NNK is in the bound form. At least a portion of thematrix-bound form co-localises with lignin—such as lignified tissue.Methods for measuring free nitrosamine(s) and matrix-boundnitrosamine(s) are well known in the art and described herein. Briefly,aliquots of tobacco samples can be extracted and the nitrosamine contenttherein can be analysed using ultra performance liquidchromatography-tandem mass spectrometry (UPLC-MS/MS). Typically, one ormore standards corresponding to the one or more nitrosamines that arebeing quantified will be incorporated into the aliquots of the tobaccosamples. The sample concentration calculated from the extractcorresponds to the “free nitrosamine(s)” concentration in the sample.After treating the extraction mixtures to the methods described herein(for example, by heating to about 130° C. for about 4 hours) nitrosamineconcentrations can again be measured by UPLC-MS/MS. From these values,the “total NNK” concentration in the samples can be calculated. The“matrix-bound NNK” concentration is the difference between the “totalNNK” and the “free NNK” concentration.

Much research has been performed on tobacco, especially in relation totobacco-specific nitrosamines. Freshly harvested tobacco leaves arereferred to as “green tobacco” and are believed to contain nonitrosamines, but green tobacco is not suitable for human consumption.The process of curing green tobacco depends on the type of tobaccoharvested. For example, Virginia flue (bright) tobacco is typicallyflue-cured, whereas Burley and certain dark strains are usuallyair-cured. The flue-curing of tobacco typically takes place over aperiod of five to seven days compared to one to two months forair-curing. Many major chemical and biochemical changes occur during thecuring process and continue through the early phases of leaf drying.

The conversion of the tobacco from its yellow to brown colour generallyresults in formation and substantial accumulation of nitrosamines,including NNK, and an increased microbial content. The exact mechanismby which tobacco-specific nitrosamines, including NNK, are formed is notclear, but is believed to be enhanced by microbial activity, involvingmicrobial nitrate reductases in the generation of nitrite during thecuring process.

As described above, matrix-bound NNK has been found to co-localise withlignin in tobacco plants or in tobacco plant material. Lignin is thegeneric term for a large group of aromatic polymers resulting from theoxidative combinatorial coupling of 4-hydroxyphenylpropanoids. Thesepolymers are deposited predominantly in the walls of secondarilythickened cells—such as fibers and tracheary elements—making them rigidand impervious. The mechanical rigidity of lignin strengthens thesetissues so that the tracheary elements can endure the negative pressuregenerated from transpiration without collapse of the tissue. In additionto providing mechanical strength, lignin has protective functions. Forexample, the physical toughness and chemical durability of lignin maydeter feeding by herbivores. Lignification is a frequent response toinfection or wounding, which may provide a physical barrier to block thepenetration of pathogens. The main building blocks of lignin are thehydroxycinnamyl alcohols (or monolignols) coniferyl alcohol and sinapylalcohol, with typically minor amounts of p-coumaryl alcohol. Themonolignols are synthesized from Phe through the general phenylpropanoidand monolignol-specific pathways. Phe is derived from the shikimatebiosynthetic pathway in the plastid. Certain enzymes of the ligninbiosynthetic pathway, namely the cytochrome P450 enzymes cinnamate4-hydroxylase (C4H), p-coumarate 3-hydroxylase (C3H), and ferulate5-hydroxylase (F5H), are membrane proteins thought to be active at thecytosolic side of the endoplasmic reticulum. Although metabolicchanneling has been shown between phenylalanine ammonia-lyase (PAL) andC4H, it remains unknown whether the other pathway enzymes are also partof metabolic complexes at the endoplasmic reticulum. The units resultingfrom the monolignols, when incorporated into the lignin polymer, arecalled guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units.

Lignin is commonly found in, for example, plant midribs, plant stems orplant stalks. Thus, the material for use in the present disclosure caninclude plant midribs or plant stems or plant stalks or a mixturethereof or a combination of two or more thereof and can be removed.Lignin is located in, for example, the vascular bundle of a tobaccoplant which can be found in plant midribs, plant stems, plant stalks andthe like. The vascular bundle is composed of a plurality of relativelyhard, cellulose members closely secured together by fibrous vegetableconnecting tissue. Surrounding this fibro-vascular bundle is the cortexwhich is formed of a relatively sponge-like vegetable tissue or coveringconstituting the larger portion of the stem and the portion which iscloser in characteristics and properties to the lamina of the tobaccoleaf. Lignin is generally located in the vascular bundle. The vascularbundle is a part of the transport system in vascular plants. Thetransport itself happens in vascular tissue, which exists in two forms,the xylem and phloem. Both these tissues are present in a vascularbundle, which in addition will include supporting and protectivetissues. Of these vascular tissues, lignin is to be found only in thexylem.

The amount of lignin can be substantially reduced in plant midribs,plant stems or plant stalks and the like or a mixture or a combinationof two or more thereof. Lignin can be substantially removed from plantmidribs, plant stems or plant stalks and the like or a mixture or acombination of two or more thereof. Suitably, the vascular bundle or thexylem or a combination thereof is substantially reduced in plantmidribs, plant stems or plant stalks and the like or a mixture or acombination of two or more thereof. Suitably, the vascular bundle or thexylem or a combination thereof is substantially removed from plantmidribs, plant stems or plant stalks and the like or a mixture or acombination of two or more thereof. Suitably, lignified sclerenchymatictissue is substantially removed from plant midribs, plant stems or plantstalks and the like or a mixture or a combination of two or morethereof.

It is an advantage that matrix-bound NNK co-localises with lignin sincelignin can be readily separated from other parts of the plant or otherplant tissues. In one embodiment, the amount of lignin is reduced byseparating lignified from non-lignified tissue. For example, the outercortex can be readily separated from the vascular bundle containinglignin, thus obtaining plant material with reduced levels of lignin. Theplant material containing reduced levels of lignin or substantially nolignin can be used in the manufacture of tobacco materials or tobaccoproducts with reduced levels of matrix-bound NNK as described herein.Optionally, the separated plant material containing lignin co-localisedwith matrix-bound NNK can be discarded or used in other processes.

In one aspect, there is provided a method of reducing or decreasing theamount of matrix-bound NNK in a cured tobacco plant or in cured tobaccoplant material comprising reducing the amount of lignin therein.According to this method, a cured tobacco plant or cured tobacco plantmaterial is provided. The amount of lignin in the cured tobacco plant orthe cured tobacco plant material is reduced. The lignin can becompletely removed or partially removed. A cured tobacco plant or curedtobacco plant material is then obtained in which the amount of lignin isreduced and the amount of matrix-bound NNK is also reduced as comparedto the cured tobacco plant or the cured tobacco plant material initiallyprovided or as compared to a control.

The lignin to be completely or partially removed can be located in thevascular bundle. The lignin to be completely or partially removed can belocated exclusively in the vascular bundle. The lignin to be completelyor partially removed can be located exclusively in the vascular bundleand not the surrounding tissue. Thus, the amount of matrix-bound NNK ina cured tobacco plant or in cured tobacco plant material is reduced byreducing the amount of the vascular bundle in the cured tobacco plant orin the cured tobacco plant material.

The lignin to be completely or partially removed can be located in thexylem. The lignin to be completely or partially removed can be locatedexclusively in the xylem. The lignin to be completely or partiallyremoved can be located exclusively in the xylem and not the surroundingtissue. Thus, the amount of matrix-bound NNK in a cured tobacco plant orin cured tobacco plant material is reduced by reducing the amount ofxylem in the cured tobacco plant or in the cured tobacco plant material.

The lignin to be completely or partially removed can be located inlignified sclerenchymatic tissue. The lignin to be completely orpartially removed can be located exclusively in lignifiedsclerenchymatic tissue. The lignin to be completely or partially removedcan be located exclusively in the lignified sclerenchymatic tissue andnot the surrounding tissue—such as the outer layer of midribs. Thus, theamount of matrix-bound NNK in a cured tobacco plant or in cured tobaccoplant material is reduced by reducing the amount of the lignifiedsclerenchymatic tissue in the cured tobacco plant or in the curedtobacco plant material.

In another aspect, there is provided a method of reducing, decreasing,preventing or inhibiting the formation of matrix-bound NNK during thecuring of a tobacco plant or tobacco plant material comprising reducingthe amount of lignin therein prior to curing. At least initially, thetobacco plant or tobacco plant material can be uncured or substantiallyuncured. The method can be used to reduce, decrease, prevent or inhibitthe co-localisation of NNK with lignin that would otherwise occur duringthe subsequent curing process. According to this aspect, an uncuredtobacco plant or uncured tobacco plant material or substantially uncuredtobacco plant or substantially uncured tobacco plant material isprovided and the amount of lignin therein is reduced prior to curing orduring curing. The lignin can be completely removed or partiallyremoved. The lignin can be located in the vascular bundle. The lignincan be located exclusively in the vascular bundle. The lignin can belocated exclusively in the vascular bundle and not the surroundingtissue. Thus, the vascular bundle can be completely removed or partiallyremoved. The lignin can be located in the xylem. The lignin can belocated exclusively in the xylem. The lignin can be located exclusivelyin the xylem and not the surrounding tissue. Thus, the xylem can becompletely removed or partially removed. The lignin can be located inthe lignified sclerenchymatic tissue. The lignin can be locatedexclusively in the lignified sclerenchymatic tissue. The lignin can belocated exclusively in the lignified sclerenchymatic tissue and not thesurrounding tissue—such as the outer layer of midribs. Thus, thelignified sclerenchymatic tissue can be completely removed or partiallyremoved. The lignin can be completely removed or partially removed.After subjecting the tobacco plant or the tobacco plant material tocuring, using methods that are well known in the art, a cured tobaccoplant or cured tobacco plant material in which the amount ofmatrix-bound NNK and the amount of lignin is reduced as compared to thestarting material or as compared to a control can be obtained.

The amount of lignin in a tobacco plant or in tobacco plant material canbe reduced using various methods that are well known in the art. In onemethod, a batch of stems or the like can be moistened or soaked influid, for example, water, which causes the cortex to soften and expandor swell whilst leaving the lignin in an unexpanded state. The cortexcan be removed manually (eg. by hand) and retained and the vascularbundle containing lignin can be discarded or used elsewhere.Accordingly, the cortex (for example, the outer cortex) can be separatedfrom lignified tissue and retained and the unexpanded lignified tissuediscarded. The cortex can then be used for further tobacco processing.There is also disclosed plant cortex (for example, plant outer cortex)or expanded plant cortex (for example, the expanded plant outer cortex)in which the amount of matrix-bound NNK is below detectable levels.Accordingly, non-lignified plant tissue can be expanded in order toseparate lignified and non-lignified tissue. Suitably, the non-lignifiedtissue is selectively or preferentially expanded over the lignifiedtissue.

By way of further example, lignin can be separated using suitabledecorticating machinery. A decorticator is a machine for stripping thebark, wood and plant stalks and the like.

In another method, mechanical separation can be used. For example,soaking of the batch of stems or the like is followed by freeze drying.In another example, soaking of the batch of stems or the like isfollowed by freeze drying, grinding and sieving. Suitably, tobacco plantmaterial can be ground into a powder form using equipment and techniquesfor grinding, milling, or the like. Suitably, the tobacco plant materialis relatively dry in form during grinding or milling, using variousequipment—such as hammer mills, cutter heads, air control mills and thelike.

The plant material can be reduced in size to form particles orparticulate matter using various methods that are known in the art. Theparticles or particulate matter can be separated by size to obtainfractions with reduced levels of lignin and reduced levels ofmatrix-bound NNK. In one suitable method, plant material is treated byimpact—such as by impact with one or more objects that are harder thanthe plant material to be treated. In one embodiment, impact withmetal—such as metal balls—is used. The impact can be delivered usingvarious methods, such as shaking. For example, plant material can beimpacted with steel balls (2 steel balls, diameter 2 cm) with shaking at300 rpm for 15 minutes. The particles or particulate matter can beseparated by size using a sieve shaker into fractions of differentparticle size(s). Suitably, the average particle is greater than about0.5 millimetres, greater than about 0.85 millimetres or greater thanabout 1 millimetre. These size fractions can have reduced levels oflignin and reduced levels of matrix-bound NNK.

The plant material can be ground or milled when the moisture contentthereof is less than about 15 weight percent to less than about 5 weightpercent. The tobacco plant material can be finely ground. Finely groundtobacco material typically has a particle size of from about 30 to 600microns.

In one embodiment, the method comprises expansion, for example bycontacting with a fluid (eg. by water soaking), followed by freezedrying, which will result in the expansion of plant material —which doesnot contain lignin or contains only low levels of lignin. Lignifiedplant material will retain a higher density, higher physical strengthand a smaller particle size than the expanded plant tissue therebypermitting separation by size. In another embodiment, the methodcomprises expansion, followed by freeze drying, followed by grinding(for example, by impact as discussed above), followed by sorting (forexample, sorting by size) of the resulting fragments and selection ofthe fragments with reduced levels of lignin and reduced levels ofmatrix-bound NNK. Sieving may be used for this purpose. In anotherembodiment, the method comprises expansion, followed by grinding orcrushing or a combination thereof, (for example, by impact, as describedabove), followed by sorting by size (using sieving or based upon densityand/or mechanical strength, for example) the resulting particles andselection of the particles with reduced levels of lignin and reducedlevels of matrix-bound NNK. The different sized fractions may alsodiffer in their free-NNK content or their NNN content or their nicotinecontent or a combination of two of more thereof. Following the completeor partial removal of lignin, the plant material can optionally befurther processed for use in a tobacco product. By way of example, thismaterial can be formed into an aqueous slurry. The resulting slurry cancontain a substantial proportion of colloidal cortex particles dispersedtherein. The conversion of the tobacco cortex into an aqueous slurry canbe accomplished using a suitable type of mill—such as a ball mill or acolloid mill. The further processing of the cortex and aqueous slurry isdescribed herein.

In certain embodiments, the plant material obtained or obtainable by themethods described herein comprises, consists or consists essentially oftissue surrounding the vascular bundle or the tissue surrounding thexylem or the tissue surrounding the lignified sclerenchymatic tissue ora combination of two or more thereof with the vascular bundle or thexylem or the lignified sclerenchymatic tissue or a combination of two ormore thereof substantially absent. In certain embodiments, the plantmaterial comprises, consists or consists essentially of the tissuesurrounding the vascular bundle or the xylem or lignifiedsclerenchymatic tissue or a combination of two or more thereof andsubstantially no vascular bundle or no xylem or no lignifiedsclerenchymatic tissue or a combination of two or more thereof. Incertain embodiments, the plant material comprises, consists or consistsessentially of the tissue surrounding the vascular bundle or the xylemor the lignified sclerenchymatic tissue or a combination of two or morethereof and no vascular bundle or no xylem or no lignifiedsclerenchymatic tissue or a combination of two or more thereof. Incertain embodiments, the plant material comprises, consists or consistsessentially of the outer tobacco cortex. In certain embodiments, theplant material comprises, consists or consists essentially of the outerlayers of plant midribs.

The methods described herein may comprise one or more further steps ofmeasuring and optionally comparing the levels of free NNK ormatrix-bound NNK or a combination thereof. Methods for measuring freeNNK and matrix-bound NNK are described herein. In one embodiment, theamount of free NNK or matrix-bound NNK or a combination thereof isdetermined in a cured tobacco plant or in cured tobacco plant material.After reducing the amount of lignin in the cured tobacco plant or thecured tobacco plant material the amount of free NNK or matrix-bound NNKor a combination thereof can be measured again. The level of at leastmatrix-bound NNK can be compared with the initial starting material toascertain if the level of matrix-bound NNK has been reduced. In thisstep, the level of at least matrix-bound NNK previously measured can becompared with the level of NNK measured following the reduction of theamount of lignin. A reduction in the amount of matrix-bound NNK in thetobacco material obtained following the reduction of the amount oflignin as compared to the tobacco material initially provided isindicative that the amount of matrix-bound NNK has been reduced.

The methods described herein may comprise one or more further steps ofmeasuring and optionally comparing the levels of free NNK ormatrix-bound NNK or combinations thereof. In one embodiment, the amountof free NNK or matrix-bound NNK or a combination thereof is measured inthe uncured tobacco plant or uncured tobacco plant material. Optionally,after reducing the amount of lignin in the uncured tobacco plant or theuncured tobacco plant material prior to curing the amount of free NNK ormatrix-bound NNK or a combination thereof can be measured again. Themethod may include one or more comparison steps. By way of example, themethod may comprise the further step of comparing the level of at leastmatrix-bound NNK initially measured, as discussed above, with the levelof NNK later measured, wherein a reduction in the amount of matrix-boundNNK in the tobacco material as compared to the tobacco materialinitially provided is indicative that the amount of matrix-bound NNK inthe tobacco material is reduced.

Free NNK or matrix-bound NNK or a combination thereof can be measured atthe start of the method and/or at the end of the method and/or duringthe method. Free NNK or matrix-bound NNK or a combination thereof may bemeasured intermittently or at intervals. The intervals may be fixedintervals or random intervals. Free NNK or matrix-bound NNK or acombination thereof can measured at the end of the method to check thatthe free NNK or matrix-bound NNK or a combination thereof is presentwithin a desired amount, concentration or range.

Lignin can be covalently or non-covalently bound to lignin. A complexcomprising lignin covalently or non-covalently bound to NNK isdescribed. A plant cell, plant tissue or plant or plant materialcomprising the complex is also disclosed. A method for reducing theamount of matrix-bound NNK in a cured tobacco plant or in cured tobaccoplant material is also described comprising reducing the amount of thecomplex therein.

In a further aspect, there is provided cured plant tissue containing areduced level of lignin, as compared to control plant tissue in whichthe amount of lignin has not been reduced, and wherein the amount ofmatrix-bound NNK is about 3500 ng/g or less. The amount of matrix-boundNNK can be about 3000 ng/g or less, about 2500 ng/g or less, about 2000ng/g or less, about 2000 ng/g or less, about 1500 ng/g or less, about1000 ng/g or less or about 500 ng/g or less. Suitably, the averageparticle size of this cured plant tissue can be greater than about 0.5millimetres, greater than about 0.85 millimetres or greater than about 1millimetre. Suitably, the amount of free NNK in this cured plant tissuecan be about 330 ng/g or less, about 300 ng/g or less, about 250 ng/g orless, about 200 ng/g or less, about 150 ng/g or less, about 100 ng/g orless or about 50 ng/g or less. Suitably, the amount of NNN in this curedplant tissue can be about 1700 ng/g or less, about 1500 ng/g or less,about 1300 ng/g or less, about 1100 ng/g or less, about 1000 ng/g orless, or about 500 ng/g or less. Suitably, the amount of nicotine inthis cured plant tissue can be about 2600 μg or less, about 2300 μg orless or about 2100 μg or less. Suitably, the amount of lignin in thiscured plant tissue can be about 6.5% or less of the total dry weightcontent of the cured plant tissue, about 6% of the total dry weightcontent of the cured plant tissue, about 5% of the total dry weightcontent of the cured plant tissue, about 4% of the total dry weightcontent of the cured plant tissue or about 3% of the total dry weightcontent of the cured plant tissue.

In one embodiment, there is provided cured plant tissue containing areduced level of lignin, as compared to control plant tissue in whichthe amount of lignin has not been reduced, and wherein the amount ofmatrix-bound NNK is about 3500 ng/g or less and the average particlesize is about 0.5 mm or greater. Suitably, the amount of free NNK isabout 300 ng/g or less. Suitably, the amount of NNN is about 1700 ng/gor less. Suitably, the amount of lignin in this cured plant tissue isabout 6.4% or less of the total dry weight content of the cured planttissue. Suitably, the amount of nicotine is about 2600 μg or less.

In another embodiment, there is provided cured plant tissue containing areduced level of lignin, as compared to control plant tissue in whichthe amount of lignin has not been reduced, and wherein the amount ofmatrix-bound NNK is about 1900 ng/g or less and the average particlesize is between about 0.85 mm and about 1 mm. Suitably, the amount offree NNK is about 250 ng/g or less. Suitably, the amount of NNN is about1270 ng/g or less. Suitably, the amount of lignin in this cured planttissue is about 4.4% or less of the total dry weight content of thecured plant tissue. Suitably, the amount of nicotine is about 2300 μg orless.

In another embodiment, there is provided cured plant tissue containing areduced level of lignin, as compared to control plant tissue in whichthe amount of lignin has not been reduced, and wherein the amount ofmatrix-bound NNK is about 1600 ng/g or less and the average particlesize is greater than about 1 mm. Suitably, the amount of free NNK isabout 200 ng/g or less. Suitably, the amount of NNN is about 1100 ng/gor less. Suitably, the amount of lignin in this cured plant tissue isabout 3% or less of the total dry weight content of the cured planttissue. Suitably, the amount of nicotine is about 2100 μg or less.

The tobacco plant or the tobacco plant material that is used at thestart of the method(s) described herein can comprise or consist orconsist essentially of uncured tobacco plant or uncured tobacco plantmaterial or cured tobacco plant or cured tobacco plant material.Processes of curing tobacco—such as tobacco leaves, especially, greentobacco leaves are well known to those skilled in the art and includewithout limitation air-curing, fire-curing, flue-curing and sun-curing.The process of curing tobacco depends on the type of tobacco harvested.For example, Virginia flue (bright) tobacco is typically flue-cured,Burley and certain dark strains are usually air-cured, and pipe tobacco,chewing tobacco, and snuff are usually fire-cured. Although tobaccoplants or tobacco plant material from any type of tobacco may be used,certain types of tobacco are preferred. Particularly preferred tobaccomaterials are selected from the group consisting of: flue-Cured,Turkish, Burley, Virginia, Maryland, Oriental, or any combination of twoor more thereof. The shape of the tobacco material is in general notlimited. It can be in the form of homogenised tobacco material. Tobaccohomogenates—such as but not limited to cured tobacco homogenates—may beprepared from tobacco material using various methods known in the art,for example, the tobacco may be in a shredded, granulated, ground orpowder form. In certain embodiments, it is desirable not to begin withtobacco material in the ground or powder form since certain mechanicalseparation methods that can be used to separate lignin can requiregrinding and/or sieving steps. The tobacco material used or obtained maycomprise additives that include, but are not limited to, one or more ofthe following components as well as combinations thereof: flavourants,organic and inorganic fillers (for example, grains, processed grains,puffed grains, maltodextrin, dextrose, calcium carbonate, calciumphosphate, corn starch, lactose, manitol, xylitol, sorbitol, finelydivided cellulose, and the like), binders (for example, povidone, sodiumcarboxymethylcellulose and other modified cellulosic types of binders,sodium alginate, xanthan gum, starch-based binders, gum arabic,lecithin, and the like), colorants (for example, dyes and pigments,including caramel colouring and titanium dioxide, and the like),humectants (for example, glycerin, propylene glycol, and the like), oralcare additives, preservatives (for example, potassium sorbate, and thelike), syrups (for example, honey, high fructose corn syrup, and thelike used as flavourants), and disintegration aids (for example,microcrystalline cellulose, croscarmellose sodium, crospovidone, sodiumstarch glycolate, pregelatinized corn starch, and the like). Suchadditives are known to those having skill in the art and may be presentin amounts and in forms known in the art.

The tobacco can be formed into reconstituted tobacco. Thus, in oneembodiment, the methods described herein can be used in the preparationof reconstituted tobacco, such as reconstituted tobacco (leaf) sheets.These sheets are paper-like material that can be made from recycledtobacco fines, tobacco stems and “class tobacco”, which consists oftobacco particles generally less than 30 mesh in size that are collectedat any stage of tobacco processing. The reconstituted tobacco can bemade by extracting the soluble chemicals in the tobacco by-products,processing the leftover tobacco fibers from the extraction into a paper,and then reapplying the extracted materials in concentrated form ontothe paper. Reconstituted tobacco can generally be formed in a variety ofways. For instance, in one embodiment, band casting can be utilised toform the reconstituted tobacco. Band casting typically employs a slurryof finely divided tobacco parts and a binder that is coated onto a steelband and then dried. After drying, the sheet is blended with naturaltobacco strips or shredded and used in various tobacco products,including as a cigarette filler. Some examples of processes forproducing reconstituted tobacco are described in U.S. Pat. No.3,353,541, U.S. Pat. No. 3,420,241, U.S. Pat. No. 3,386,449, U.S. Pat.Nos. 3,760,815 and 4,674,519. Reconstituted tobacco can also be formedby a papermaking process. Some examples of processes for formingreconstituted tobacco according to this process are described in U.S.Pat. No. 3,428,053, U.S. Pat. No. 3,415,253, U.S. Pat. No. 3,561,451,U.S. Pat. No. 3,467,109, U.S. Pat. No. 3,483,874, U.S. Pat. No.3,860,012, U.S. Pat. No. 3,847,164, U.S. Pat. No. 4,182,349, U.S. Pat.No. 5,715,844, U.S. Pat. No. 5,724,998; and U.S. Pat. No. 5,765,570. Forexample, the formation of reconstituted tobacco using papermakingtechniques can involve the steps of mixing tobacco with water,extracting the soluble ingredients therefrom, concentrating the solubleingredients, refining the tobacco, forming a web, reapplying theconcentrated soluble ingredients, drying, and threshing. Variousingredients—such as flavour or colour treatments—can be applied to theweb.

The tobacco obtained or obtainable by the methods described herein maybe formed into a tobacco sheet—such as a reconstituted tobacco sheet.According to this embodiment, the method may comprise the steps of: (a)obtaining tobacco material—such as a tobacco homogenate—according to themethods described herein; (b) preparing a slurry of tobacco homogenate;(c) casting the slurry of the tobacco homogenate; and (d) drying theslurry of the tobacco homogenate to form a reconstituted tobacco sheet.According to another embodiment, the method may comprise the steps of:(a) obtaining tobacco material—such as a tobacco homogenate—according tothe methods described herein and preparing a tobacco slurry; (b) castingthe slurry of the tobacco homogenate; and (c) drying the slurry of thetobacco homogenate to form a tobacco sheet. The step of casting theslurry of the tobacco homogenate may be performed using any of thecasting or paper making processes that are known in the art. By way ofexample, casting processes are described in U.S. Pat. No. 5,724,998 andU.S. Pat. No. 5,584,306; paper-making processes are described in U.S.Pat. No. 4,341,228; U.S. Pat. No. 5,584,306 and U.S. Pat. No. 6,216,706.Casting processes typically include casting the slurry onto a continuousstainless steel belt, drying the cast slurry to form a reconstitutedtobacco sheet and removing said sheet. Paper-making processes typicallyinclude casting the aqueous slurry from a head box onto a wire screenfor forming the desired sheet. The aqueous slurry may be separated intoa soluble portion and a fibrous portion. Water is drained from thefibrous portion and a sheet is so-formed is subsequently treated anddried.

The tobacco slurries may further comprise one or more binders—such asgums and pectins. As described above, tobacco slurries that are used toprepare reconstituted tobacco sheets may further comprise commonadditives that include, but are not limited to, one or more of thefollowing components as well as combinations of these: wood cellulosefibers, aerosol formers, sugars, and flavourants and binders. Additivesof the list described above are known to those having skill in the artand may be present in these aqueous slurries in amounts and in formsknown in the art.

Once prepared, the reconstituted tobacco sheets described herein may becut in a similar fashion as whole leaf tobacco to produce tobacco fillersuitable for cigarettes and other tobacco products. The reconstitutedtobacco sheets described herein may be further trashed or flayed withmechanical fingers into sized pieces similar to natural tobacco laminastrips or cut into diamond shaped pieces, between about 50 to 100 mm ona side. The reconstituted tobacco sheet pieces described herein may befurther blended with other tobaccos such as flue-cured tobacco, Burleytobacco, Maryland tobacco, Oriental tobacco, rare tobacco, specialtytobacco, expanded tobacco and the like. The precise amount of each typeof tobacco within a tobacco blend used for the manufacture of aparticular cigarette brand varies from brand to brand. See, for example,Tobacco Encyclopaedia, Voges (Ed.) p. 44-45 (1984), Browne, The Designof Cigarettes, 3rd Ed., p. 43 (1990) and Tobacco Production, Chemistryand Technology, Davis et al. (Eds.) p. 346 (1999). The entire blend maythen be shredded into a cut filler and incorporated into a tobaccoproduct.

According to a further aspect, there is provided a method for blendingtobacco in which at least two different types of tobacco are blended soas to form a tobacco blend. The various tobacco blends have differentrecipes for blending different tobacco types. Tobacco types can be, byway of example, Burley, Flue Cured, Oriental, Bright and Reconstitutedtobacco. Burley, Flue Cured and Oriental tobacco are specific types oftobacco, while Bright tobacco is a pre-blend of Flue Cured and Orientaltobacco. According to the method, a first (type of) cured tobacco plantmaterial is provided and the amount of lignin therein is reduced. Any ofthe methods described herein can be used to reduce the amount of lignin.The total and/or matrix-bound NNK content of the first cured tobaccoplant material can be measured and cured tobacco plant material in whichthe total and/or matrix-bound NNK content is reduced as compared tofirst cured tobacco plant material initially provided can be selectedfor further use. A second cured tobacco plant material which has ahigher total and/or matrix-bound NNK content than the total and/ormatrix-bound NNK of the first cured tobacco plant material is nextprovided. In some embodiments, the total and/or matrix-bound NNK contentof this material may already be known so measurement of these valueswill not be required. In other embodiments, the total and/ormatrix-bound NNK content of this material may not be known and someasurement will be required. Thus, measuring the total and/ormatrix-bound NNK content in the second cured tobacco plant material isan optional step in this method. The first and second cured tobaccoplant materials obtained from these steps can be blended together usingprocesses that are well known in the art. Optionally, the total and/ormatrix-bound NNK content in the final blended tobacco plant material canbe measured. According to this method, a blended tobacco plant materialcan be obtained in which the total and/or matrix-bound NNK content ofthe final blended tobacco plant material is lower than the second curedtobacco plant material. Advantageously, this method can be used toprovide a blend of tobacco material in which the overall NNK content ofthe blend is reduced. Essentially, the tobacco material in which theamount of lignin therein has been reduced is used to dilute or reducethe overall NNK content in the blended tobacco material.

The tobacco material obtained or obtainable according to this disclosurecan also be used in tobacco cut filler and in a smoking article formedfrom a tobacco rod of the cut filler. Conventionally, cut filler tobaccoproducts for smoking articles are formed predominantly from the laminaportion of the tobacco leaf, which is separated from the stem portion ofthe leaf during a threshing process. Much of the stem portion thatremains after the lamina has been removed and separated is not used. Inorder to increase the amount of the tobacco material that can be usedcommercially, some tobacco stems can be added back into the cut fillertogether with the lamina. In order to improve the taste and burningcharacteristics of the tobacco stem for use in the cut filler, the stemsare often first subjected to one or more treatment procedures, which caninclude the procedures described herein. The rolling step can be carriedout on tobacco stems that have been subjected to the method of thepresent disclosure. The stems can be rolled to a desired thickness—suchas a mean thickness of about 0.6 mm to 0.8 mm. During subsequentprocessing and storage steps, the stems can expand to a final thicknessof about 0.8 mm to about 1.0 mm. After rolling, the stems are dried andtransferred to the tobacco production plant, where they are cut andadded to the tobacco cut filler. In some cases, the rolling step mayalternatively be incorporated as part of the on-line production processfor cut filler. Typically the moisture content of the tobacco stems isabout 28% to about 34% oven volatiles prior to rolling in order toprevent damage to the structure of the stems. If necessary, the tobaccostems can be conditioned prior to rolling in order to increase themoisture content to this level. Known processes for conditioning tobaccostems involve contacting the stems with water, steam or a mixture ofwater and steam. In methods where the rolling step is incorporatedon-line and dried stems are used, the conditioning step will typicallytake longer and may require a soaking step in which the stems are soakedin water for a number of hours prior to rolling. The tobacco stems canbe rolled using a one-step rolling process to reduce the thickness ofthe stems to the desired mean thickness. After rolling, the stems can becut to a cut width of between 0.1 mm and 0.2 mm. The cut rolled stemsare then optionally expanded using known stem expansion techniques, andthen dried. Where the stems are pre-rolled and dried, it will typicallybe necessary to condition the stems prior to cutting in order toincrease the moisture content of the tobacco stems back to between 28%and 34% oven volatiles. This increases the pliability of the tobaccostems in order to limit damage or breakage of the stems during cutting.Finally, the cut rolled stems are combined with tobacco cut lamina andany additional tobacco materials in order to form cut filler having atleast 5% by weight of the cut rolled tobacco stems. Thus, in a furtheraspect, there is provided a method for preparing tobacco for use as atobacco cut filler comprising the steps of: (a) performing the method(s)as described herein; and (b) rolling and cutting the tobacco materialfor use as a tobacco cut filler. There is also described a method oftreating tobacco material—such as tobacco stems—for use in tobacco cutfiller, the method comprising the steps of: (a) performing the method asdescribed herein; (b) rolling the tobacco material; (c) cutting therolled tobacco material; and (d) optionally drying the cut rolled stems.The rolled tobacco stems can be combined with tobacco lamina such thatthe steps are carried out on the combined tobacco stems and lamina. Thecutting step can comprise cutting the rolled stems to a cut width ofbetween about 0.3 mm and 1.3 mm. The method can comprise the steps of:removing stems from the tobacco leaf; cutting the stems to an averagelength of between about 15 mm and 80 mm; and rolling the stems to athickness of between 0.1 mm and 0.5 mm. A method of producing cut fillercomprising rolled tobacco stems is also provided, the method comprising:treating tobacco stems using the method described herein; and blendingthe treated stems with at least one type of tobacco lamina, expandedtobacco or reconstituted tobacco to produce cut filler. The tobacco cutfiller obtained or obtainable by this method can comprise at least 60%,and preferably at least 80% by weight tobacco lamina having a mean cutwidth between 0.8 mm and 1.1 mm, suitably, about 0.9 mm, and a meanthickness of about 0.2 mm. The tobacco cut filler can comprise up to 95%by weight tobacco lamina with a mean cut width between about 0.8 mm and1.1 mm, more suitably about 0.9 mm, and a mean thickness of about 0.2mm. The particles of tobacco lamina in the cut filler are therefore ofsimilar dimensions to the particles of tobacco stem. As such, thetobacco stems are not visually distinct from the tobacco lamina, even ata high inclusion rate. In addition, the blend of tobacco stems andlamina can advantageously be transported and processed effectivelywithout significant settling of the stems. Suitably, the mean cut widthof the cut rolled tobacco stems is within about 0.1 mm, more suitablywithin about 0.05 mm of the mean thickness of the tobacco lamina in thecut filler. Cut fillers may be incorporated into a variety of smokingarticles. For example, the cut filler may be used in the tobacco rod ofa combustible smoking article, such as a filter cigarette, cigarillo orcigar. Alternatively, the cut filler may be used to provide the tobaccoaerosol generating substrate in a distillation based smoking article, oran electrically heated smoking system. Alternatively, the cut filler maybe used as a roll-your-own product, or loose tobacco product forexample, for use in a pipe.

The tobacco material can be incorporated into various consumableproducts—such as tobacco products. Also encompassed are methods formaking such tobacco products. Tobacco products include withoutlimitation smoking articles or smokable articles and smokeless tobaccoproducts, including non-combustible products, heated products, andaerosol-generating products. Non-limiting examples of smoking orsmokable articles include cigarettes, cigarillos, cigars and pipetobaccos. Non-limiting examples of smokeless tobacco products includechewing tobaccos, snuffs, and substrates for use in aerosol-generatingproducts. Smokeless tobacco products may comprise tobacco in any form,including as dried particles, shreds, granules, powders, or a slurry,deposited on, mixed in, surrounded by, or otherwise combined with otheringredients in any format, such as flakes, films, tabs, foams, or beads.Liquid contents of smokeless tobacco products can be contained in adevice or enclosed in a form, such as beads, to preclude interactionwith a water-soluble wrapper. The wrapper may be shaped as a pouch topartially or completely enclose tobacco-incorporating compositions, orto function as an adhesive to hold together a plurality of tabs, beads,or flakes of tobacco. Exemplary materials for constructing a wrapperinclude film compositions comprising HPMC, CMC, pectin, alginates,pullulan, and other commercially viable, edible film-forming polymers.Other wrapping materials may include pre-formed capsules produced fromgelatin, HPMC, starch/carrageenan, or other commercially availablematerials. Such wrapping materials may include tobacco as an ingredient.Wrappers that are not orally disintegrable may be composed of woven ornonwoven fabrics, of coated or uncoated paper, or of perforated orotherwise porous plastic films. Wrappers may incorporate flavouring orcolouring agents. Smokeless products can be assembled together with awrapper utilizing any method known to persons skilled in the art ofcommercial packaging, including methods such as blister packing, inwhich a small package can be formed by a vertical form/fill/sealpackaging machine.

The amount of matrix-bound NNK in these smokable articles, smokelessproducts and aerosols and the like may be at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, and 100% lower—such as about 200% or 300%lower—when compared to consumable products derived from control tobaccoplant material. The amount of free-NNK may be substantially unchanged.

The amount of matrix-bound NNK in these smokable articles, smokelessproducts and aerosols and the like may be about 3500 ng/g or less, orabout 3000 ng/g or less, or about 2500 ng/g or less, or about 2000 ng/gor less, or about 1500 ng/g or less, or about 1000 ng/g or less, orabout 500 ng/g or less.

The amount of free NNK in these smokable articles and smokeless productsand the like may be about 330 ng/g or less, about 300 ng/g or less,about 250 ng/g or less, about 200 ng/g or less, about 150 ng/g or less,about 100 ng/g or less or about 50 ng/g or less.

The amount of NNN in these smokable articles, smokeless products andaerosols and the like may be about 1700 ng/g or less, about 1500 ng/g orless, about 1300 ng/g or less, about 1100 ng/g or less, about 1000 ng/gor less, or about 500 ng/g or less.

The amount of nicotine in these smokable articles and smokeless productsand the like may be about 2600 μg or less, about 2300 μg or less, orabout 2100 μg or less, or about 2000 μg or less 1900 μg or less, orabout 1800 μg or less.

The amount of lignin in these smokable articles and smokeless productsand the like may about 6.5% or less of the total dry weight content ofthe cured plant tissue, about 6% of the total dry weight content of thecured plant tissue, about 5% of the total dry weight content of thecured plant tissue, about 4% of the total dry weight content of thecured plant tissue or about 3% of the total dry weight content of thecured plant tissue.

In one embodiment, the amount of matrix-bound NNK in these smokablearticles and smokeless products and the like is about 3500 ng/g or less,the amount of free NNK is about 300 ng/g or less, the amount of NNN isabout 1700 ng/g or less, the amount of lignin is about 6.4% or less ofthe total dry weight content of the cured plant tissue, and the amountof nicotine is about 2600 μg or less.

In one embodiment, the smokable articles or smokeless products and thelike comprise about 3500 ng/g or less of matrix-bound NNK. Suitably, theamount of free NNK is about 300 ng/g or less. Suitably, the amount ofNNN is about 1700 ng/g or less. Suitably, the amount of lignin in isabout 6.4% or less of the total dry weight content of the cured planttissue. Suitably, the amount of nicotine is about 2600 μg or less.Suitably, the average particle size is about 0.5 mm or greater. Inanother embodiment, the smokable articles or smokeless products and thelike comprise about 1900 ng/g or less matrix-bound NNK. Suitably, theamount of free NNK is about 250 ng/g or less. Suitably, the amount ofNNN is about 1270 ng/g or less. Suitably, the amount of lignin in thiscured plant tissue is about 4.4% or less of the total dry weight contentof the cured plant tissue. Suitably, the amount of nicotine is about2300 μg or less. Suitably, the average particle size is between about0.85 mm and about 1 mm.

In another embodiment, the smokable articles or smokeless products andthe like comprise about 1600 ng/g or less matrix-bound NNK. Suitably,the amount of free NNK is about 200 ng/g or less. Suitably, the amountof NNN is about 1100 ng/g or less. Suitably, the amount of lignin inthis cured plant tissue is about 3% or less of the total dry weightcontent of the cured plant tissue. Suitably, the amount of nicotine isabout 2100 μg or less. Suitably, the average particle is greater thanabout 1 mm.

The tobacco material can be derived from tobacco plants, which includeplants of the genus Nicotiana, various species of Nicotiana, includingN. rustica and N. tabacum. The tobacco material can be derived fromvarieties of Nicotiana species, commonly known as flue or brightvarieties, Burley varieties, dark varieties and oriental/Turkishvarieties. In some embodiments, the tobacco material is derived from aBurley, Virginia, flue-cured, air-cured, fire-cured, Oriental, or a darktobacco plant. In some embodiments, the tobacco material is derived, forexample, from one or more of the following varieties: N. tabacum AA37-1, N. tabacum B 13P, N. tabacum Xanthi (Mitchell-Mor), N. tabacum KTD#3 Hybrid 107, N. tabacum Bel-W3, N. tabacum 79-615, N. tabacum SamsunHolmes NN, F4 from cross N. tabacum BU21×N. tabacum Hoja Parado, line97, N. tabacum KTRDC#2 Hybrid 49, N. tabacum KTRDC#4 Hybrid 1 10, N.tabacum Burley 21, N. tabacum PM016, N. tabacum KTRDC#5 KY 160 SI, N.tabacum KTRDC#7 FCA, N. tabacum KTRDC#6 TN 86 SI, N. tabacum PM021, N.tabacum K 149, N. tabacum K 326, N. tabacum K 346, N. tabacum K 358, N.tabacum K 394, N. tabacum K 399, N. tabacum K 730, N. tabacum KY 10, N.tabacum KY 14, N. tabacum KY 160, N. tabacum KY 17, N. tabacum KY 8959,N. tabacum KY 9, N. tabacum KY 907, N. tabacum MD 609, N. tabacum McNair373, N. tabacum NC 2000, N. tabacum PG 01, N. tabacum PG 04, N. tabacumP01, N. tabacum P02, N. tabacum P03, N. tabacum RG 11, N. tabacum RG 17,N. tabacum RG 8, N. tabacum Speight G-28, N. tabacum TN 86, N. tabacumTN 90, N. tabacum VA 509, N. tabacum AS44, N. tabacum Banket A1, N.tabacum Basma Drama B84/31, N. tabacum Basma I Zichna ZP4/B, N. tabacumBasma Xanthi BX 2A, N. tabacum Batek, N. tabacum Besuki Jember, N.tabacum 0104, N. tabacum Coker 319, N. tabacum Coker 347, N. tabacumCriollo Misionero, N. tabacum PM092, N. tabacum Delcrest, N. tabacumDjebel 81, N. tabacum DVH 405, N. tabacum Galpao Comum, N. tabacumHB04P, N. tabacum Hicks Broadleaf, N. tabacum Kabakulak Elassona, N.tabacum PM102, N. tabacum Kutsage E1, N. tabacum KY 14xL8, N. tabacum KY171, N. tabacum LA BU 21, N. tabacum McNair 944, N. tabacum NC 2326, N.tabacum NC 71, N. tabacum NC 297, N. tabacum NC 3, N. tabacum PVH 03, N.tabacum PVH 09, N. tabacum PVH 19, N. tabacum PVH 2110, N. tabacum RedRussian, N. tabacum Samsun, N. tabacum Saplak, N. tabacum Simmaba, N.tabacum Talgar 28, N. tabacum PM132, N. tabacum Wislica, N. tabacumYayaldag, N. tabacum NC 4, N. tabacum TR Madole, N. tabacum PrilepHC-72, N. tabacum Prilep P23, N. tabacum Prilep PB 156/1, N. tabacumPrilep P12-2/1, N. tabacum Yaka JK-48, N. tabacum Yaka JB 125/3, N.tabacum TI-1068, N. tabacum KDH-960, N. tabacum TI-1070, N. tabacumTW136, N. tabacum PM204, N. tabacum PM205, N. tabacum Basma, N. tabacumTKF 4028, N. tabacum L8, N. tabacum TKF 2002, N. tabacum TN90, N.tabacum GR141, N. tabacum Basma xanthi, N. tabacum GR149, N. tabacumGR153, and N. tabacum Petit Havana. The use of any species of the genusNicotiana is disclosed, including N. rustica and N. tabacum (forexample, LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart1000-1, and Petico). Other species include N. acaulis, N. acuminate, N.acuminate var. multiflora, N. alata, N. amplexicaulis, N. arentsii, N.benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola,N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior,N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N.gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N.langsdorffii, N. linearis, N. longffiora, N. megalosiphon, N. miersii,N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N.occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora,N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N.repanda, N. rosulata, N. rosulata subsp. ingulba, N. rotundifolia, N.setchellii, N. simulans, N. solanifolia, N. spegazzinii, N. stocktonii,N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N.tomentosiformis, N. trigonophylla, N. umbratica, N. velutina, N.wigandioides, and N. x sanderae.

The use of tobacco cultivars and elite tobacco cultivars is alsocontemplated herein. Particularly useful Nicotiana tabacum varietiesinclude Burley type, dark type, flue-cured type, and Oriental typetobaccos. Non-limiting examples of varieties or cultivars are: BD 64, CC101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973,HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC,K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200,KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14xL8LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow LeafMadole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100,NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD207, PD 7302 LC, PD 7309 LC, PD 7312 LC′ ‘Periq'e’ tobacco, PVH03,PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81,RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, SpeightG-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (TomRosson) Madole, VA 309, VA359, AA 37-1, B 13P, Xanthi (Mitchell-Mor),Bel-W3, 79-615, Samsun Holmes NN, KTRDC number 2 Hybrid 49, Burley 21,KY 8959, KY 9, MD 609, PG 01, PG 04, P01, P02, P03, RG 11, RG 8, VA 509,AS44, Banket A1, Basma Drama B84/31, Basma I Zichna ZP4/B, Basma XanthiBX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero,Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf,Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, RedRussian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, PrilepHC-72, Prilep P23, Prilep PB 156/1, Prilep P12-2/1, Yaka JK-48, Yaka JB125/3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002,GR141, Basma xanthi, GR149, GR153, Petit Havana. Low convertersubvarieties of the above, even if not specifically identified herein,are also contemplated.

The following examples are provided as an illustration and not as alimitation. Unless otherwise indicated, the present invention employsconventional techniques and methods of molecular biology and plantbiology.

EXAMPLES Example 1 Method for Analysis of Free and Matrix-Bound NNK inTobacco

Aliquots of tobacco samples (for example, about 750 mg) are extractedwith about 30 mL of Tris-HCl buffer (50 mM; pH 7.4) by shaking for aboutone hour at approximately room temperature. Internals standard (100ng/mL NNK-d₄) are added. Samples (0.4 mL) of the extracts are filteredusing a 0.2 μM filter and the NNK content is analysed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).The sample concentrations calculated from these extract concentrationscorrespond to the “free NNK” concentrations in the sample. Aftertreating the extraction mixtures (for example, by heating to about 130°C. for about 4 hours) and filtering aliquots of the extracts, NNKconcentrations are again measured by UPLC-MS/MS. From these values, the“total NNK” concentration in the samples can be calculated. The“matrix-bound NNK” concentration is the difference between the “totalNNK” and the “free NNK” concentrations.

An alternative method for “total-NNK” extraction comprises acidificationof the extraction mixtures with concentrated HCl (for example, 3 mL of37% HCl added to 30 mL) and incubation for 48 hours at 80° C. The acidicextracts are neutralised before filtration and UPLC analysis by addingNaOH solution (6N, 40 μL) and magnesium hydroxide suspension (10%; 40μL) to 320 μL of extract.

Example 2 UPLC Analysis

The column used is Waters Acquity BEH C18, 1.7 μm, 2.1×50 mm. Theeluents used are: (A) ammonium bicarbonate (10 mM; adjusted to pH 9.8with ammonia)+2% (v/v) acetonitrile; (B) acetonitrile. The gradient usedis 0 min—5% B; 0.5 min—5% B; 3.3 min—18.3% B. The flow that is used is0.5 mL/min. The column temperature that is used is 50° C.

Example 3 MS/MS Methodology

This analysis is carried out on a Waters TQ spectrometer using thefollowing MRM transitions: NNK: 208.2→122.2; dwell time 100 ms; NNK-d4:212.2→126.2; dwell time 100 ms; Capillary voltage: 0.6 kV; Cone voltage:25 V; Collision energy: 11 eV; Source temperature: 120° C.; Desolvationtemperature: 400° C.; Desolvation gas flow: 800 L/h.

Example 4 Distribution of Matrix-Bound NNK in Lignified andNon-Lignified Tissues of Burley Stems

About 2 grams of midribs of cured Burley tobacco leaves are separated byhand into inner lignified tissue (36% of total dry weight) and outernon-lignified tissue (64% of total dry weight). In each of these samplesthe concentration of free NNK and total NNK is analysed by UPLC-MS, asdescribed above. Matrix-bound NNK is calculated as the differencebetween free NNK and total NNK concentration. Lignin content isquantified using a photometric method based on derivatisation withthioglycolic acid (see Brinkmann et al. (2002) J. Chem. Ecol., 28,2483-2501).

The results in FIG. 1 show the distribution of free NNK, matrix-boundNNK and lignin in lignified (L) and non-lignified (NL) tissues of curedBurley stems. FIG. 2 is a cross-section of a hydrated cured Burley stemshowing lignified (L) and non-lignified (NL) tissues. Lignified tissueis stained red with phloroglucinol. The results in FIG. 3 shows thematrix-bound NNK content of lignified and non-lignified tissues of greenmidribs after nitrosating with sodium nitrite solution.

These results show that matrix-bound NNK is distributed principally inlignified tissues of Burley tobacco stems and midribs.

Example 5 Enrichment of a Fraction with Low Bound-NNK Content fromBurley Stem by Freeze-Drying, Grinding and Size Separation

A sample of Burley Stems (52 g) is humidified with water (350 mL) andfreeze-dried. A part of the resulting material (12 g) is ground byshaking with steel balls (2 balls, diameter 2 cm; 300 rpm; 15 min) andseparated with a sieve shaker into fractions of different particle sizeranging from greater than 1 mm to less than 0.25 mm.

FIG. 4 and Table 1 show the free- and matrix-bound NNK, NNN, lignin andnicotine levels in sieving fractions of ground freeze-dried Burleystems. The analysis of free- and matrix-bound NNK in FIG. 5 and Table 1indicates that both the lignin content (as % dry weight of eachfraction) and the matrix-bound NNK content is decreased in the fractionswith particle size of greater than 0.5 mm, including fractions withparticle size of 0.5 mm to 0.85 mm, 0.85 mm to 1 mm and greater than 1mm. FIG. 5 shows that lignin content correlates well with matrix-boundNNK thereby confirming the co-localisation of lignin and matrix-boundNNK.

Example 6 Localization of Matrix-Bound NNK Precursor in Green TN90Midribs and Burley Stems

The relative distribution of matrix-bound NNK is measured insclerenchymatic and non-sclerenchymatic tissue of TN90 midribs. Theprediction of matrix-bound NNK being bound to lignin predicts a higherconcentration of this precursor in the lignified sclerenchymatic tissue.In a second experiment the relative distribution of free andmatrix-bound NNK in sclerenchymatic and non-sclerenchymatic tissue of(cured) Burley stems is investigated.

Materials & Methods Green Midribs

The midribs (only the proximal halves) of 15 mature TN90 leaves aremanually separated in sclerenchymatic tissue (S) (the “center” of themidrib) and non-sclerenchymatic tissue (NS). Both are freeze-dried andfinely ground. The water-insoluble fraction of the two materials isdetermined by extracting 1 g, each, three times with 40 mL ofmethanol/water 1:3 (room temperature for 1 hour) and weighing theinsoluble material (designated SW and NSW) after freeze-drying.Pseudo-oxynictoine (PON) and nicotine analysis in S and NS (n=5) ismeasured using the follow methods. Finely powdered plant material (˜20mg) is extracted by shaking at room temperature for 45 minutes withmethanol/water (4:1) containing PON-methyl-d₃ as an internal standard(200 ng/mL). After filtration (0.2 μm) samples are subjected to LC-MSanalysis using the following conditions: Column: Acquity UPLC BEH C18column (1.7 μm, 50×2.1 mm; Waters); Column temperature: 50° C.; Eluents:Aqueous ammonium bicarbonate adjusted to pH 9.8 using NH₃ withacetonitrile (98:2, v/v; eluent A); acetonitrile (eluent B); Gradient: 0min—0% B, 0.5 mL/min; 0.5 min—0% B, 0.5 mL/min; 6 min—30% B, 0.5 mL/min;MS detection: PON m/z 179.2 à m/z 106.1; PON-methyl-d₃ m/z 182.2 à m/z106.1; UV detection: 260 nm. PON and nicotine are eluted after 2.6 and4.1 minutes, respectively. For the quantification of nicotine the peakarea at 260 nm and external calibration is used. In order to estimatethe content of matrix-bound NNK precursor in S, NS, SW and NSW, aliquots(˜20 mg; n=5) of these materials are nitrosated by incubation inNaNO2-solution (1.5 mL (10 mg/mL in water)) for 4 hours at roomtemperature with shaking, centrifuged and washed/centrifuged four timeswith 10 mL water. Then, the centrifugation sediment of each nitrosatedsample is taken up in 4 mL Tris-HCl buffer (50 mM pH 7.5; with NNK-d4and NNN-d4 at 100 ng/mL), autoclaved (for 4 hours at 130° C.) andanalysed for NNK content using the methods described herein.

Cured Burley Stems

A Burley stem sample (2 g=four ˜5 cm pieces) is manually separated intothe outer (non-lignified) tissue (CNS) and the inner (lignified) part(CS). Both samples are finely ground in a mixer mill (Retsch“Tissuelyzer” for 2.5 minutes, 50 s-1). This results in 1183 mg and 651mg of CNS and CS powder, respectively. Free NNK in the two samples isdetermined after extraction of ˜50 mg aliquots (n=5) with 1.5 mLTris-buffer (+IS) for 1 hour at room temperature. Total NNK isdetermined after autoclave extraction (130° C. for 4 hours) of ˜50 mgaliquots (n=5) in 5 mL Tris-buffer (+IS).

Results

The results of this experiment are shown in Table 2 and in FIG. 6. Afternitrosation and washing, a 7-fold higher concentration of matrix-boundNNK is found in the sclerenchymatic tissue (S) compared to the outerlayers of the midribs (NS). PON and nicotine are both two-fold higher inNS than in S. The results of these artificial nitrosation experimentsare corroborated by the bound-NNK levels in the lignified (CS) andnon-lignified (CNS) parts of a commercially cured Burley stem sample.While free NNK is two-fold higher in CS, matrix-bound NNK is 7-foldhigher in CS. Nicotine and NNN levels are higher in CS than CNS and lessthan NNK.

CONCLUSIONS

The presence of high-concentrations of matrix-bound NNK precursor in thelignified, sclerenchymatic tissue of green midribs indicates thatmatrix-bound NNK is covalently or non-covalently linked to lignin.

Any publication cited or described herein provides relevant informationdisclosed prior to the filing date of the present application.Statements herein are not to be construed as an admission that theinventors are not entitled to antedate such disclosures. Allpublications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in cellular, molecular and plant biology or related fieldsare intended to be within the scope of the following claims.

TABLE 1 free Fraction of total weight NNK NNN NIC bound NNK LigninSieving fraction [%] [ng/g] [ng/g] [ug/g] [ng/g] [% d.w.] A: >1 mm 16200 1098 2091 1606 2.92 A: 0.85-1 mm 4 251 1269 2307 1905 4.41 A:0.5-0.85 mm 16 328 1690 2608 3500 6.33 A: 0.25-0.5 mm 27 463 2305 29376218 11.31 A: <0.25 mm 38 297 1732 3237 2936 4.81 total 329 1760 28393651 6.47

TABLE 2 dry after water- water- fresh dry mat- before wash- insolublesinsolubles weight weight ter washing ing [% dry [% fresh [g] [g] [%][mg] [mg] weight] weight] S 25 3.1 12.4 1010 668 66 8.2 NS 171 11 6.41010 414 41 2.6 CS 0.65 CNS 1.18 PON/ NNK After Free PON NIC NIC *autoclaving NNN NNK Sample [ug/g] [ug/g] 1000 [ng/g] [ng/g] [ng/g] S 26513245 1660 NS 116 184 111 SW 331 17610 1595 NSW 109 326 272 PON-S 6.8784 8.63 PON-NS 14.0 1909 7.32 CS-free 9496 1356 120 CNS-free 7985 89564 CS-total 8459 2038 CNS-total 7057 310

1. A method of reducing the amount of matrix-bound NNK in cured tobaccoplant material comprising separating lignified tissue from non-lignifiedtissue, wherein the amount of lignin is reduced mechanically.
 2. Themethod according to claim 1, comprising the steps of: (a) providingcured tobacco plant material; (b) separating lignified fromnon-lignified tissue in the cured tobacco plant material; and (c)obtaining cured tobacco plant material in which the amount of lignin isreduced and the amount of matrix-bound NNK is reduced as compared to thecured tobacco plant material provided in step (a).
 3. The methodaccording to claim 2, wherein following step (a) there is a further stepof measuring the amount of at least matrix-bound NNK, and optionally,wherein following step (b) there is a further step of measuring theamount of at least matrix-bound NNK.
 4. The method according to claim 3,wherein said method comprises the further step (d) of comparing thelevel of at least matrix-bound NNK measured following step (a) with thelevel of matrix-bound NNK measured following step (b), wherein areduction in the amount of matrix-bound NNK in the tobacco materialobtained in step (b) as compared to the tobacco material provided instep (a) is indicative that the amount of matrix-bound NNK in thetobacco material is reduced.
 5. A method of reducing the formation ofmatrix-bound NNK during curing of tobacco plant material comprisingreducing the amount of lignin therein prior to curing, comprising thesteps of: (a) providing uncured tobacco plant material; (b) mechanicallyseparating lignified from non-lignified tissue in the uncured tobaccoplant material prior to curing; (c) curing the tobacco plant materialprovided in step (b); and (d) obtaining cured tobacco plant material inwhich the amount of matrix-bound NNK is reduced as compared to a controlin which the amount of lignin has not been reduced.
 6. The methodaccording to claim 5, wherein following step (a) there is a further stepof measuring the amount of at least matrix-bound NNK, and optionally,wherein following step (b) there is a further step of measuring theamount of at least matrix-bound NNK and optionally, wherein followingstep (c) there is a further step of measuring the amount of at leastmatrix-bound NNK, preferably, wherein following step (c) or step (d)said method comprises the further step of comparing the level of atleast matrix-bound NNK measured following step (a) with the level of atleast matrix-bound NNK measured following step (b) and/or step (c),wherein a reduction in the amount of matrix-bound NNK in the tobaccomaterial obtained in step (b) or step (c) as compared to the tobaccomaterial provided in step (a) is indicative that the amount ofmatrix-bound NNK in the tobacco material is reduced.
 7. The methodaccording to claim 1, wherein the tobacco plant material is treated toexpand non-lignified plant tissue, preferably, wherein the amount oflignin is reduced by separating the expanded and non-expanded planttissue based on their different densities and/or their differentstrengths and/or their different particle sizes and/or wherein theamount of lignin is reduced by removing at least the vascular bundle orxylem or lignified sclerenchymatic tissue or a combination of two ormore thereof from the plant material; and/or wherein the plant materialprovided in step (a) comprises or consists or consists essentially ofplant midribs or plant stems or plant stalks or a combination of two ormore thereof.
 8. Tobacco plant material obtained or obtainable by themethod according to claim
 1. 9. A method for manufacturing a tobaccoproduct having reduced levels of matrix-bound NNK, comprising:mechanically separating lignified tissue from non-lignified tissue intobacco to reduce lignin as compared to control tobacco plant material,forming the tobacco product from the non-lignified tissue, wherein saidlevels of matrix-bound NNK are reduced as compared to the control.
 10. Amethod for producing reconstituted tobacco comprising the steps of: (a)performing the method according to claim 1; (b) manufacturing thetobacco material obtained in step (a) into reconstituted tobacco; and(c) optionally incorporating the reconstituted tobacco into a tobaccoproduct.
 11. Reconstituted tobacco obtained or obtainable by the methodof claim
 10. 12. A method for preparing tobacco for use as a tobacco cutfiller comprising the steps of: (a) performing the method according toclaim 1; and (b) rolling and cutting the tobacco material for use as atobacco cut filler.
 13. Cured tobacco plant material containing areduced level of lignin as compared to control tobacco plant material inwhich the amount of lignin has not been reduced, wherein the amount oflignin in the cured tobacco plant material has been reduced bymechanically separating lignified tissue from non-lignified tissue, andwherein the amount of matrix-bound NNK is about 3500 ng/g or less,preferably, wherein the amount of free NNK is less than about 330 ng/g,optionally wherein the NNN content is less than about 1700 ng/g andoptionally wherein the nicotine content is less than about 2610 μg/g;and/or wherein the cured tobacco plant material comprises, consists ofconsists essentially of plant cortex—such as outer plant cortex; and/orwherein vascular bundle or xylem or lignified sclerenchymatic tissue ora combination thereof is substantially absent from the cured planttissue.
 14. A tobacco product or a reconstituted tobacco productcomprising, consisting or consisting essentially of the tobacco plantmaterial according to claim
 8. 15. A method for blending tobacco inwhich at least two different types of tobacco are blended so as to forma tobacco blend comprising the steps of: (a) providing a first curedtobacco plant material and reducing the amount of lignin therein bymechanically separating lignified tissue from non-lignified tissue; (b)measuring the total and/or matrix-bound NNK content of the first curedtobacco plant material and selecting cured tobacco plant material inwhich the total and/or matrix-bound NNK content is reduced as comparedto the first cured tobacco plant material provided in step (a); (c)providing a second cured tobacco plant material which has a higher totaland/or matrix-bound NNK content than the total and/or matrix-bound NNKof the first cured tobacco plant material obtained in step (b), andoptionally measuring the total and/or matrix-bound NNK content in thesecond cured tobacco plant material; (d) blending together the first andsecond cured tobacco plant materials from steps (b) and (c) andoptionally measuring the total and/or matrix-bound NNK content in theblended tobacco plant material; and (e) obtaining a blended tobaccoplant material in which the total and/or matrix-bound NNK content of theblended tobacco plant material is lower than the second cured tobaccoplant material provided in step (c), optionally wherein steps (a) and(b) are performed after step (c).
 16. A tobacco product or areconstituted tobacco product comprising, consisting or consistingessentially of the tobacco plant material according to claim 13.